The application of a column packing material, 3-(l,8-naphthalimido)propyl-modified silyl silica gel (NAIP) developed by us, for high performance liquid chromatography (HPLC) of barbiturates and diastereomeric compounds is described. The separation of barbiturates by using an NAIP column as a stationary phase was at first investigated. Six commercially available barbiturates (Barbital, Phenobarbital, Amobarbital, Pentobarbital, Secobarbital and Thiopental) were well separated on an NAIP column by an isocratic elution with borate buffer (pH 7.0)-acetonitrile (9:1, v/v). Further, the applicability of the HPLC system including an NAIP column to the determination of barbiturates in human blood plasma was examined. Barbiturates extracted from plasma sample using a solvent extraction (hexane-diethyl ether, 3:7. v/v) were subjected to the HPLC system and determined by an internal standard method with UV detection at 220 nm. The recoveries of barbiturates spiked to human plasma were obtained in the range of 88.6 to 100.0%. The lower detection limits of barbiturates spiked ranged from 0.05 to 0.33 fig/mL, and the within-day and day-to-day precisions for plasma sample (n=5) gave relative standard deviations of less than 9 07% and 18.68%, respectively.